In the context of the LIPS assay, the test antigens must be fused with a luciferase protein to enable the efficient detection of the antibodyCantigen complexes [29]. group died within 14 days post-challenge. Serum samples collected on 41 days post-vaccination were analyzed for their reactivity against a panel of 29 viral structural proteins. The sera of pigs from the LAV group exhibited a strong antibody reactivity against various viral structural proteins, while the sera of pigs in the KV group only displayed weak antibody reactivity against the inner envelope (p32, p54, p12). There was a negative correlation between the intensity of antibody reactivity against five ASFV antigens, namely p12, C-178 p14, p15, p32, and pD205R, and the viral DNA titers in the blood of animals after the challenge infection. Thus, antibody reactivities against these five antigens warrant further evaluation as potential indicators of vaccine-induced protection. Keywords: African swine fever virus, live attenuated vaccine, inactivated vaccine, antibody profile, humoral immunity, luciferase-based immunoprecipitation system 1. Introduction African swine fever virus (ASFV) is the causative agent of a hemorrhagic disease in pigs, with Has2 the mortality rate often approaching 100%. Over the past few years, the virus has spread to many countries in Asia, Europe, and Central America, causing substantial economic losses [1]. ASFV is a large double-stranded DNA virus belonging to the genus in the family (sequence. As a result, the ASFV protein is separated from the protein by a C-178 flexible linker. The resulting plasmids were sequenced to verify the authenticity of the ASFV genes. To generate Nluc-tagged antigens, the plasmids were transfected into HEK-293T cells as previously described [28]. At 60 h post-transfection, the cells were collected and lysed in the RIPA lysis buffer (ThermoFisher Scientific, Rockford, IL, USA), supplemented with 1 protease inhibitor (Pierce Protease Inhibitor Tablet, EDTA-Free, ThermoFisher Scientific, Rockford, IL, USA). The cell lysates were centrifuged at 17,000 for 10 min, and the supernatant was passed through a 0.45 mm filter C-178 to remove insoluble cell debris. The cell extracts were stored in small aliquots at ?80 C for future use. 2.7. Luciferase-Immunoprecipitation System The luciferase-immunoprecipitation system (LIPS) was performed as previously described [28,29]. The overview of the LIPS assay is depicted in Figure 1. Briefly, test serum samples were diluted 1:40 in buffer A (50 mM Tris, pH 7.5, 100 mM NaCl, 5 mM MgCl2, 1% Triton X-100, Rohm & Haas, Philadelphia, PA, USA), and passed through a 0.45 m filter to eliminate huge aggregates. Fifty L of every diluted sera had been transferred to a proper of the 96-well dish and blended with 50 L from the Nluc-tagged antigen remove containing around 107 comparative light systems (RLUs). Each ASFV antigen was examined in duplicate. This real way, the antibody reactivity of 1 serum sample could be examined with 47 ASFV antigens in a single 96-well dish, using a control antigen jointly. The dish was incubated for 1 h at area temperature on the rocking system. Ten L of proteins A Sepharose 4B (Invitrogen, Camarillo, CA, USA) pre-washed and diluted in 50 uL of buffer A was put into each well from the dish. After another 1 h incubation, the complete content in the 96-well dish was used in a 96-well filtration system HTS dish (EMD Millipore, Billerica, MA, USA) for cleaning on vacuum pressure manifold. Following the last clean, Nano-Glo? Luciferase substrate (Promega, Madison, WI, USA) was put into each well. The luminescence sign was assessed using the SpectraMax L audience (Molecular Gadgets, San Jose, CA, USA). For every work, a swine serum test filled with antibodies against the Nluc proteins was used being a positive control. Likewise, a pool of serum examples gathered from ASFV-negative pigs was utilized as a poor control..